Hyperhomocysteinemia HHcy is a clinical condition characteri

Hyperhomocysteinemia (HHcy) is a clinical condition characterized by increased levels of plasma homocysteine (Hcy) and a well-known risk factor for CVDs. Hcy is a sulfur-containing non-protein amino Bleomycin A5 hydrochloride formed during the intracellular conversion of methionine to cysteine. Previous study demonstrated that HHcy impaired autophagy in primary astrocytes (Tripathi et al., 2016). But effect of HHcy on autophagy in VSMCs remains elusive. In addition, our previous study found that Hcy could up-regulate ETB receptors in VSMCs (Chen et al., 2016a; Chen et al., 2016b). However, it is unclear whether autophagy is involved in Hcy-induced-up-regulation of ETB receptor in VSMCs. Based on these reports, our hypothesis is that Hcy up-regulated ETB receptors via suppression of autophagy in VSMCs. The present study was designed to test our hypothesis. It may provide us with a new perspective for mechanism of Hcy-induced CVDs.
Materials and methods
Results
Discussion The present study showed that Hcy up-regulated the levels of beclin-1 protein expression and the ratios of LC3B II/LC3B I, reduced number of LC3B positive puncta, down-regulated the levels of p62 protein expression in SMA, which suggested that Hcy could inhibit autophagy in SMA. Moreover, Hcy also increased levels of ETB receptor protein expression, and enhanced ETB receptor-mediated contractile responses. This indicted that up-regulation of ETB receptor was associated with inhibition of autophagy in SMA. Furthermore, AICAR and Rap not only elevated Hcy-inhibited levels of autophagy, but also inhibited Hcy-increased levels of ETB receptor via activation of AMPK and inhibition of mTOR, respectively. Moreover, MHY1485 recovered AICAR-decreased levels of ETB receptor in Hcy group in SMA. These data suggested that Hcy up-regulated ETB receptor by inhibiting autophagy in VSMCs via AMPK/mTOR signaling pathway. Autophagy was divided into three main types: Macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy is thought to be the major form of autophagy and herein will be referred to as “autophagy”. The molecular activation of autophagy is primed via phosphorylation of ULK1(Atg1), which then coordinates interactions of other critical proteins in the autophagy cascade, leading to encapsulation of cellular constituents in a double-membrane vesicle called the autophagosome. The autophagosome then fuses with the lysosome, leading to degradation of the compartmentalized contents and release of essential building blocks such as amino acids for reutilization (He et al., 2003). Autophagy in VSMCs plays an important role in the progress of vascular disease (Grootaert et al., 2015). During autophagy, the cytosolic form of microtubule associated LC3-I is converted to the phosphatidylethanolamine-conjugated form of LC3 (LC3-II) to promote the formation of autophagosome, which stabilizes the growing autophagosome. Formation of LC3-II is typically used as an indication of autophagic activation (He et al., 2003). In present study, there were high protein express levels of LC3B II in culture SMA. This suggested that autophagy occurred in culture SMA. However, Hcy decreased organ culture-up-regulated the ratios of LC3B II/LC3B I. In addition, Hcy also reduced number of LC3B positive puncta. This suggested that Hcy impaired autophagy in VSMCs. The scaffolding adaptor protein p62 interacts with both LC3II and polyubiquitinated protein, which leads to the self-degradation as well as degradation of polyubiquitinated proteins in autolysosomes. The activation of an autophagic flux can reduce the content of p62. Thus, the level of p62 can indicate whether autophagy is completed (Komatsu et al., 2007). In present study, Hcy significantly increased the levels of p62 compared with organ culture. This suggested that Hcy inhibited autophagic flux. Beclin-1 is the mammalian homolog of yeast Atg6. Beclin-1 complexes are associated with initiating the formation of autophagic vacuoles. Thus, beclin-1 is considered a reliable method for detecting autophagy flux (Yue et al., 2003). In present study, compared to organ culture, Hcy suppressed the levels of beclin-1, which suggested that Hcy blocked the formation of autophagic vacuoles in VSMCs.